Urethritis is a common complaint among men seeking primary care, but its etiology and long-term consequences are incompletely understood. Urethritis of any etiology can augment HIV shedding; urethritis is often associated with either single or multiple known sexually transmitted infections (STI) that can be transmitted to women, who in turn are at risk for reproductive complications. Common etiologies of urethritis include sexually transmitted bacteria, with Neisseria gonorrhoeae (NG) causing gonococcal urethritis (GU), and Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), and probably Ureaplasma urealyticum (UU) causing nongonococcal urethritis (NGU). Other bacteria, viruses (adenoviruses, HSV) and the protozoan Trichomonas vaginalis (TV) occasionally cause NGU. However, no pathogen is detected in 25-50% of men with the NGU subset of urethritis; this group is defined as having idiopathic urethritis (IU). The appropriate management and treatment of IU is unknown. The goal of this project is to seek microbial etiologies for IU using modern metagenomic techniques, bioinformatics and statistical approaches applied to a large, carefully defined and described population of men with IU, men with no urethritis, and men with IU and NGU who have clinical treatment failure. We examine our overarching hypothesis, that the composition of the urethral microbiome is crucial to development of IU, in 3 Specific Aims. We hypothesize that some cases of IU are: 1) associated with previously unidentified pathogens (Aim 1); 2) associated with high loads of organisms not otherwise thought to be urethral pathogens (Aim 2); and 3) not associated with specific organisms, but rather with broad microbial community shifts known as dysbiosis. The project constitutes a definitive evaluation of potential microbial associations of IU.